The Effects Of Thalidomide On Proliferation, Collagen Synthesis, The Expression Of MMP-1and TIMP-1mRNA In Cultured Human Dermal Fibroblasts In Vitro

Objective：Dermal fibroblasts are the main cellular components in thedermis and play an important role in the synthesis and secretion ofextracellular matrix and fibers. They are extremely important to theoccurrence of the normal composition of dermal connective tissue, skin woundhealing and dermal fibrotic diseases. Skin fibrosis is an excessiveaccumulation of extracellular matrix phenomenon which is due to an abnormalproliferation of fibroblast and the overproduction of collagen that is difficult tobe degraded. Fibrotic diseases include the involvement of the multiple organfibrosis disease such as idiopathic pulmonary fibrosis, liver cirrhosis,scleroderma, renal fibrosis, etc. Their pathogenesis are unknown and havedifferent clinical manifestations, however they have a common characteristic:excessive deposition of extracellular matrix (mainly collagen) in the damagedtissue. More and more experiments prove that thalidomide can inhibit fibrosisof animal model and fibrosis of person’s lung, liver, heart, and other organs.For example, it has been reported that thalidomide can inhibit the pulmonaryinterstitial fibrosis in mice by inhibiting the proliferation of lung fibroblastsand collagen synthesis. In recent years, although many studies have reportedthe effect of thalidomide on fibroblasts of animal，human lung，synovial, bonemarrow etc, but the effect of thalidomide on normal human skin fibroblastshas not yet been reported.The purpose of the experiment is to observe the effects of thalidomide onproliferation, collagen synthesis, the expression of MMP-1andTIMP-1mRNA in cultured human dermal fibroblasts in vitro. Aims to explorethe mechanism of action of thalidomide on human dermal fibroblasts andprovide a reliable theoretical basis for the possibility of clinical application of skin fibrosis diseases.Methods:1Human dermal fibroblast was cultured in vitro by enzyme digestionmethod and was appraised by immunohistochemistry. The fifth generationcells were selected to experiment.2Using MTT assay to detect the proliferation of fibroblasts among thedifferent thalidomide concentration groups（10-3mol/L～10-11mol/L）, find outthe effective concentration of thalidomide to fibroblasts, so as to group thefollowing experiment.3Established the experimental group and the control group according tothe results of MTT assay, the Hydroxyproline kit was used to detect thecollagen content of fibroblasts.4According to the result of MTT, establishing the experimental andcontrol group, extracting total RNA of each group, assessing the integralityand content of RNA, the level of MMP-1mRNA and TIMP-1mRNAexpression were detected by reverse transcriptase-polymerase chain reaction（RT-PCR） in the Human dermal fibroblasts cells treated before and afterwith thalidomide.5Statistical analysis: The experimental results were analyzed bystatistical methods with SPSS15.0software.First the date were analyzed bytest its normality. The data which haven’t conformed Gaussian distributionwere expressed in quartile or median and analyzed by Mann-Whitney test.Thedata that have conformed Gaussian distribution were expressed inmean±Standard deviation and analyzed by ANOVA. P＜0.05consideredstatistically significant.6Each experimental procedure was operated strictly on basis ofinstructions.Results:1MTT assay showed： Within the concentration of (10-3mol/L～10-6mol/L) thalidomide can obviously inhibit proliferation of Human dermalfibroblasts cells in vitro,There was significantly statistical significance of inhibition in different concentration groups of thalidomide compared with thecontrol group (P＜0.05), Concentration of10-3mol/L and10-4mol/L showedclear inhibitory effect, compared with groups10-5mol/L and10-6mol/L,(P＜0.05). But the difference of proliferation between the control group and10-7～10-11mol/L thalidomide groups had no statistical significance(P＞0.05)2The effect of thalidomide on content of hydroxyproline in thesupernatant：The level of hydroxyproline was lower in different concentration groupsof thalidomide（10-4,10-5,10-6,10-7mol/L）compared to control group, there wassignificant differences (P＜0.05). The level of hydroxyproline was lowest inthe10-4mol/L group among the all experimental groups(P＜0.05).3The expression of MMP-1mRNA before and after thalidomide t-reatment was determined by using RT-PCR:In the experimental groups, the expression of MMP-1mRNA wasincreased in different concentrations of thalidomide（10-4,10-5,10-6,10-7mol/L）compared to control group. And there were significant differences(P＜0.05).Also there were significant differences between groups when they werecompared in pairs(P＜0.05).4The expression of TIMP-1mRNA before and after thalidomidetreatment was determined by using RT-PCR.In the experimental groups, the expression of MMP-1mRNA wasdecreased in different concentrations of thalidomide（10-4,10-5,10-6,10-7mol/L）compared to control group. And there were significant differences(P＜0.05).Also there were significant differences between groups when they werecompared in pairs(P＜0.05).Conclusion:1We confirmed that thalidomide had the ability of inhibiting theproliferation of Human dermal fibroblasts cells in vitro.2Thalidomide can inhibit the synthesis of collagen.3Within a certain drug concentration, thalidomide can up-regulation theexpression of MMP-1mRNA but down-regulated the expression of TIMP-1 mRNA in Human dermal fibroblasts cells.4Thalidomide may be due to its inhibition of human dermal fibroblastproliferation activity, collagen synthesis, TIMP-1expression and thepromotion of MMP-1expression, to provide reliable basis guidance in theapplication of skin fibrosis disease and further research.